Trafficking 1 MCB 110 - Spring 2008- Nogales 4 MEMBRANE TRAFFICKING
I Introduction: secretory pathway A. Protein Synthesis and sorting B. Methods to study cytomembranes
II Endoplasmic Reticulum A. Smooth ER B. Rough ER C. Synthesis of proteins in membrane-bound ribosomes • The signal hypothesis • Synthesis of membrane proteins D. Glycosylation in the ER
III Golgi IV Vesicle Transport A. COPII-coated Vesicles B. COPI-coated Vesoicles C. Clathrin-coated Vesicles V Lisosomes A. Phagocytosis B. Autophagy VI Endocytosis
Suggested Reading: Lodish, Chapter 5, 5.3; Chapter 16, 16.1 to 16.3; Chapter 17 Alberts, Chapters 12 and 13 Trafficking 2 MCB 110 - Spring 2008- Nogales
I Introduction to the Secretory Pathway
The eukaryotic cell is filled with membranous organelles that form part of an integrated and dynamic system shuttling material across the cell Trafficking 3 MCB 110 - Spring 2008- Nogales
The biosynthetic or secretory pathway includes the synthesis of proteins in the ER, their modification in the ER and Golgi, and their transport to different destinations such as the plasma membrane, lysosomes, vacuoles, etc.
In constitutive secretion materials are transported in a continual manner. In regulated secretion , materials are stored in secretory granules in the periphery of the cell and discarded in response to a particular stimulus (e.g. nerve cells, cells producing hormones or digestive enzymes).
Materials are transported in vesicles that move along microtubules, powered by motor proteins. Sorting is facilitated by receptors localized in particular membranes. Trafficking 4 MCB 110 - Spring 2008- Nogales Methods to Study Cytomembranes
Visualization by electron microscopy Dynamic localization by autoradiography and pulse-chase Trafficking 5 MCB 110 - Spring 2008- Nogales Trafficking 6 MCB 110 - Spring 2008- Nogales
Use of GFP constructs
Movement of proteins through the secretory pathway has been followed using a Green Flourescence Protein (GFP). Cells where infected with vesicular stomatitis virus (VSV) in which one of the genes (VSVG) is fused to GFP. Large amounts of VSVG protein are produced in the ER that move to the Golgi and then to the plasma membrane. The process can be seen as a wave of green fluorescence that can be synchronized using temperature mutants of VSVG than cannot leave the ER at high temperatures. Trafficking 7 MCB 110 - Spring 2008- Nogales Subcellular fraction purification and characterization: Differential Centrifugation & Cell Fractionation.
When cells are homogenized the rough ER breaks up into small closed vesicles call microsomes. Trafficking 8 MCB 110 - Spring 2008- Nogales
Cell-Free Systems
Inmediately after their synthesis, secretory proteins are localized in the lumen of microsomes.
If proteases are added to the microsomes, the secretory protein is not digested.
If the microsomes are treated with detergent previous to their exposure to proteases, the secretory protein is digested. Trafficking 9 MCB 110 - Spring 2008- Nogales
Mutant Studies
Yeast cells: they are small, fast growing and haploid
Yeast continuously secrete a number of proteins, one of them is invertase. Temperature-sensitive mutants strains have been identified where the secretion of proteins is block at the non-permissive temperature. These are called sec mutants. The analysis of these mutants identified 5 classes (A-E) that correspond to 5 steps in the secretory pathway with distinctive distribution of cytoplasmic membranes. Trafficking 10 MCB 110 - Spring 2008- Nogales Trafficking 11 MCB 110 - Spring 2008- Nogales Protein Targeting Trafficking 12 MCB 110 - Spring 2008- Nogales Trafficking 13 MCB 110 - Spring 2008- Nogales Trafficking 14 MCB 110 - Spring 2008- Nogales Endoplasmic Reticulum Smooth ER Lacks ribosomes Tubular Membranes Functions: M Synthesis of steroid hormones Detoxification of the liver Secuestration of Ca2+ (skeletal muscle) SER RER Rough ER Ribosomes on the cytosolic side Flattened stacks Functions: Synthesis of secretory proteins Granule • Intestinal cells: mucoproteins • Endocrine cells: polypeptide hormones • Plasma cells: antibodies • Liver cells: blood serum proteins Synthesis of membrane proteins Synthesis of soluble, endomembrane proteins Post-translational modification Protein folding/control Trafficking 15 MCB 110 - Spring 2008- Nogales Trafficking 16 MCB 110 - Spring 2008- Nogales Synthesis of Proteins on Membrane-bound Ribosomes
The Signal Sequence Hypothesis Trafficking 17 MCB 110 - Spring 2008- Nogales Trafficking 18 MCB 110 - Spring 2008- Nogales Electron Microscopy and 3-D Reconstruction
General Applicability
No crystallization is required Applicable to very large complexes Requires very small amounts of sample
Study of Fully Assembled, Functional Complexes
- In near physiological conditions - In different functional states - Structural Basis of Function and Regulation Trafficking 19 MCB 110 - Spring 2008- Nogales Imaging and Reconstruction of Biological Macromolecules
EM + Noise
3D 2D Averaging
Volume (3D) Projection (2D) Experimental Image Trafficking 20 MCB 110 - Spring 2008- Nogales SRP-Ribosome Structure Trafficking 21 MCB 110 - Spring 2008- Nogales
Experimental prove of the cotranslational insertion of secretory proteins into the ER. Trafficking 22 MCB 110 - Spring 2008- Nogales
Experimental identification of proteins that form the translocon. Trafficking 23 MCB 110 - Spring 2008- Nogales Trafficking 24 MCB 110 - Spring 2008- Nogales Translocon-Ribosome Structure Trafficking 25 MCB 110 - Spring 2008- Nogales Trafficking 26 MCB 110 - Spring 2008- Nogales Synthesis of Integral Membrane Proteins Trafficking 27 MCB 110 - Spring 2008- Nogales Trafficking 28 MCB 110 - Spring 2008- Nogales Trafficking 29 MCB 110 - Spring 2008- Nogales Trafficking 30 MCB 110 - Spring 2008- Nogales
Post-translational modifications and quality control in the RER
Both the soluble and membrane proteins synthesized in the ER undergo several modifications before they move to Golgi:
Formation of disulfide bonds – This modification can occur in the ER but not in the cytosol. Although the enzyme that catalyzes the reaction has not been identified, the redox environment in the ER is more appropriate for the oxidation of sulphydryl groups (-SH).
Folding – The proper folding of new proteins and the assembly of subunits into multimeric proteins is facilitated by several ER proteins. Only those proteins that are properly folded can progress to the Golgi, those misfolded or unassembled are transported to the cytosol where they are degraded.
Addition and processing of carbohydrates – Glycosylation of many plasma-membrane and secretory proteins is initiated in the ER and continues in the Golgi. Trafficking 31 MCB 110 - Spring 2008- Nogales
II D – Glycosylation in the ER • Most proteins synthesized in the ER become glycosylated. • The sequence of sugars in the oligosaccharide are highly specific •Carbohydrates are important for interaction with other macromolecules. They also confer extra stability to many extracellular glycoproteins. • Addition of sugars is catalyzed by membrane-bound enzymes called glycosyltransferases.
Oligosaccharides can be added to a protein through an asparagine (N- linked). This occurs both in the ER and the Golgi. N-linked oligosaccharides tend to be large and branched. Synthesis starts by the addition of a large precursor to the protein.
They can also be added via a serine or a threonine (O-linked). This occurs only in the Golgi. O-linked oligosaccharides tend to be short, one to four sugars in length. Sugars are added one at a time. Trafficking 32 MCB 110 - Spring 2008- Nogales
Production of N-linked Oligosaccharides
•The core of the oligosaccharide is assembled on a carrier molecule, dolichol phosphate.
• 1-9: sugars are added one at a time, with the first steps taking place in the cytosol and the rest in the ER cisternae. The donor is always a nucleotide:
CMP-sialic acid GDP-mannose UDP-N-acetylglucosamine
10: A finally assembled block of 14 sugars is transferred by oligosaccharidetransferase to an asparagine residue in the protein as is being translocated into the ER.
11-13: Dolichol phosphate is recycled for a new round of assembly
Modification of this core oligosaccharide starts in the ER with the removal of the terminal glucose residues by glucosidases. Trafficking 33 MCB 110 - Spring 2008- Nogales Trafficking 34 MCB 110 - Spring 2008- Nogales
III Golgi
Formed by flattened, disk-like cisternae with dilated rims, associated tubules and vesicles. Functional compartments: • Cis Golgi network (CGN) - Sorting of proteins between ER and Golgi. • Cis, medial, trans cisternae - sequential protein modification: • proteolytic cleavage • amino acid modifications • Carbohydrates modifications • Trans Golgi network (TGN) - Sorting of vesicles to different destinations Trafficking 35 MCB 110 - Spring 2008- Nogales
Differential staining of Golgi compartments
Osmium tetraoxide in Antibodies for Mannosidase II in Antibodies for diphosphatase cis cisternae Medial cisternae in Trans cisternae Trafficking 36 MCB 110 - Spring 2008- Nogales
Models of Transport within the Golgi There are two opposing models on how materials move through the Golgi system.
• In the maturation model each cis cisterna matures into a transcisterna. The evidence comes from cells that produce secretory products too large to fit into vesicles. • In the alternative model cisternae stay in place and movement occurs through vesicles budding from one cisterna and fusing into the next. The evidence comes from reconstituted vesicle transport experiments in cell-free systems.
Two populations of Golgi-derived membrane fractions were prepared from cultured cells. The “donor” fraction was from cells infected with VSV, a virus that encodes an integral membrane protein VSV-G that is synthesized in the ER and glycosylated in the Golgi of infected cells. These donor cells were mutants that lacked N-acetylglucosamine transferase, which is normally in the medial cisternae. A “acceptor” fraction was obtained from uninfected, wild type cells.
Only when the two fractions were mixed did VSV-G become glycosylated. This was monitored by using radioactively labeled N-acetylglucosamine. Trafficking 37 MCB 110 - Spring 2008- Nogales Trafficking 38 MCB 110 - Spring 2008- Nogales
Golgi Functions O-linked glycosylation and completion of N-linked glycosilation are carried out in the Golgi. The latter includes the removal of mannose residues and the attachment of other types of monosaccharides. Trafficking 39 MCB 110 - Spring 2008- Nogales
IV Vesicle Transport
In the biosynthetic pathway materials are carried by vesicles (50-75 nm) that bud from donor membranes and fuse to acceptor membranes.
Importantly, the assymetry of the membrane is maintained throughout this trip in the endomembrane system of the cell. Trafficking 40 MCB 110 - Spring 2008- Nogales Trafficking 41 MCB 110 - Spring 2008- Nogales Trafficking 42 MCB 110 - Spring 2008- Nogales Trafficking 43 MCB 110 - Spring 2008- Nogales Trafficking 44 MCB 110 - Spring 2008- Nogales
Most vesicles are covered by a protein coat on the cytoplasmic surface and are referred to as coated vesicles. The functions of the coat are: • To act as mechanical devices that force the membrane to curve and form a budding vesicle. • To select the membrane and soluble cargo to be carried by the vesicle.
There are three main types of coated vesicles: • COPII-coated vesicles - From the ER to the Golgi. • COPI-coated vesicle - From Golgi back to the ER (and retrogade within the Golgi). • Clathrin-coated vesicles - From TGN to final destination (endosomes, lysosomes,…); in the endocytic pathway. Trafficking 45 MCB 110 - Spring 2008- Nogales
IV A - COPII-coated vesicles
These vesicles were identified in yeast mutants deficient in transport from ER to Golgi. Later in mammals antibodies against homologous proteins block budding of ER vesicles, with no effect in other vesicles.
• There are five COPII proteins • Sar1 is a GTP-binding protein that when activated binds to the membrane and nucleates the assembly of the coat. • Tansmembrane cargo receptors bind the soluble cargo in the lumen of the ER and recruit the COPII proteins by interaction through their signal sequences on the cytosolic side. •Interaction of Sar with the receptor-bound COPII proteins results in the formation of a coated bud and eventually in a coated vesicle. • Disassembly of the coat before fusion is thought to occur by hydrolysis of the GTP in Sar, decreasing his affinity for the vesicle. Trafficking 46 MCB 110 - Spring 2008- Nogales Trafficking 47 MCB 110 - Spring 2008- Nogales Model of Sar1 mechanism Trafficking 48 MCB 110 - Spring 2008- Nogales
IV B - COPI-coated vesicles
• There are eight COPI proteins. • ARF protein is a GTP-binding protein. Hydrolysis is required for disassembly of the coat. • They are involved in bulk flow (non specific) and selective transport. • Required for the recovery of ER proteins in retrograde transport. Trafficking 49 MCB 110 - Spring 2008- Nogales
Retrieving ER proteins • ER proteins are maintained in the ER by retention in large complexes that cannot be transported in vesicles, and by active retrieval. • Specific retrieval signal in the C-terminus of proteins (KDEL) bind to specific receptors in the Golgi that are incorporated into COPI vesicles and returned to the ER • Similar mechanisms are likely to exists for other compartments. Trafficking 50 MCB 110 - Spring 2008- Nogales
Targeting
Vesicle targeting is mediated in all eukaryotes by the SNARE family of proteins. SNARES have been best characterized in nerve cells. The plasma membrane of the presynaptic nerve cell contains the protein syntaxin, which binds specifically to the protein VAMP in the membrane of synaptic vesicles.
VAMP-like proteins (v-SNARES) are localized in the membrane of transport vesicles. Syntaxin-like proteins (t-SNARES) are situated in the target membrane. Targeting is dictated by the specific interaction of corresponding v-SNARES and t-SNARES.
In addition to SNARES there is a number of proteins involved in the docking process, including Rab, SNAPs and NSF. Trafficking 51 MCB 110 - Spring 2008- Nogales Trafficking 52 MCB 110 - Spring 2008- Nogales Trafficking 53 MCB 110 - Spring 2008- Nogales Trafficking 54 MCB 110 - Spring 2008- Nogales Trafficking 55 MCB 110 - Spring 2008- Nogales Trafficking 56 MCB 110 - Spring 2008- Nogales IV C - Clathrin-coated vesicles
Final protein sorting occurs in the TGN, from which clathrin-coated vesicles form. Clathrin-coated vesicles contain: • An outer structural scafold or clathrin lattice. • An inner shell of adaptors, protein complexes covering the cytosolic surface of the vesicle.
Adaptors bind on the outside to clathrin, and on the inside to membrane receptors. The receptors bind specifically to the soluble ligands in the cysternae. Formation of the clathrin scaffold mechanically favors the budding of the vesicles. Once budded the vesicle sheds its cathrin coat and moves to its destination where it fuses to the receiving membrane. Trafficking 57 MCB 110 - Spring 2008- Nogales V Lysosomes • Membrane organelles containing hydrolytic enzymes (up to 50) produced in the ER. They work at acid pH of 4.6. • In the lysosome the pH is maintained by the H+-ATPase. The lysosomal membranes is protected from attack by highly glycosylated integral membrane proteins. • Lysosomes have variable appearance and size (1 m to 25 nm).
Phagocytosis - Uptake of particulate material by single-cell organisms and phagocytes (e.g. macrophages). • Mycobacterium tuberculosis inhibits fusion of phagosome and lysosome • Listeria monocytogenes (meningitis) has a phospholipase that destroys the lisosome membrane.
Autophagy - Destruction of the cell’s own organelles. The organelle (e.g. mitochondrion) is enveloped by ER membrane and then fuses to a lysosome. Trafficking 58 MCB 110 - Spring 2008- Nogales
Sorting of Lysosomal proteins
Unlike other proteins in the TGN, lisosomal proteins have a phosphorylated mannose group (added in the cis-Golgi) that acts as a recognition signal that binds specifically to a membrane receptor enriched in clathrin-coated vesicles. Trafficking 59 MCB 110 - Spring 2008- Nogales Trafficking 60 MCB 110 - Spring 2008- Nogales
VI Endocytosis
Endocytic Pathway - Starts with a dynamic system of tubules and vesicles called endosomes: early endosomes are near the plasma membrane, where soluble materials are send forward in the pathway and membrane proteins are sent back for recycling: and late endosomes, which are closer to the nucleus. The two classes differ in density, pH, and protein composition.
Bulk-phase endocytosis is the non-specific uptake of extracellular fluids. It is most common in cells that are converting plasma membrane into cytosolic membrane.
Receptor-mediated endocytosis requires the binding of the ligand to a receptor on the outside of the plasma membrane. In the plasma membrane receptors concentrate in areas called coated pits, surrounded in the cytosolic side by a clathrin network. Trafficking 61 MCB 110 - Spring 2008- Nogales Trafficking 62 MCB 110 - Spring 2008- Nogales Trafficking 63 MCB 110 - Spring 2008- Nogales
Each clathrin molecule consists of 3 heavy chains and three light chains forming a triskelion.
The triskelion is the assembly unit for the formation of polygonal lattices. These lattices become curved and eventually form a vesicle during budding, by conversion of hexagoms into pentagons. Trafficking 64 MCB 110 - Spring 2008- Nogales Trafficking 65 MCB 110 - Spring 2008- Nogales
+ Light Chains Cryo-Electron Microscopy 3-D Reconstructions Trafficking 66 MCB 110 - Spring 2008- Nogales
Dynamin - GTP-binding protein required for the formation of endocytic vesicles. It self-assembles into rings and helices that form a collar around the neck of the invaginated coated pit. GTP hydrolysis is thought to induce a conformational change in the helix that constricts the neck and pinches off the vesicle. Trafficking 67 MCB 110 - Spring 2008- Nogales Trafficking 68 MCB 110 - Spring 2008- Nogales EXAMPLE: LDL Trafficking 69 MCB 110 - Spring 2008- Nogales Trafficking 70 MCB 110 - Spring 2008- Nogales